www.nature.com/scientificreports OPEN The Fate of a Hapten - From the Skin to Modifcation of Macrophage Migration Inhibitory Factor (MIF) in Received: 14 September 2017 Accepted: 31 January 2018 Lymph Nodes Published: xx xx xxxx Isabella Karlsson 1, Kristin Samuelsson2, Carl Simonsson2, Anna-Lena Stenfeldt2, Ulrika Nilsson1, Leopold L. Ilag1, Charlotte Jonsson2 & Ann-Therese Karlberg2 Skin (contact) allergy, the most prevalent form of immunotoxicity in humans, is caused by low molecular weight chemicals (haptens) that penetrate stratum corneum and modify endogenous proteins. The fate of haptens after cutaneous absorption, especially what protein(s) they react with, is largely unknown. In this study the fuorescent hapten tetramethylrhodamine isothiocyanate (TRITC) was used to identify hapten-protein conjugates in the local lymph nodes after topical application, as they play a key role in activation of the adaptive immune system. TRITC interacted with dendritic cells but also with T and B cells in the lymph nodes as shown by fow cytometry. Identifcation of the most abundant TRITC-modifed protein in lymph nodes by tandem mass spectrometry revealed TRITC-modifcation of the N-terminal proline of macrophage migration inhibitory factor (MIF) – an evolutionary well-conserved protein involved in cell-mediated immunity and infammation. This is the frst time a hapten-modifed protein has been identifed in lymph nodes after topical administration of the hapten. Most haptens are electrophiles and can therefore modify the N-terminal proline of MIF, which has an unusually reactive amino group under physiological conditions; thus, modifcation of MIF by haptens may have an immunomodulating role in contact allergy as well as in other immunotoxicity reactions. About 20% of the population in the Western world have skin (contact) allergy to one or more compounds in their close environment1. Allergic contact dermatitis (ACD), i.e. skin infammation and eczema, is the clinical manifestation of contact allergy and afects 5–10% of the population2,3. ACD is caused by T-lymphocyte mediated type IV hypersensitivity responses to antigens afer skin exposure to contact allergens. Small (<1000 Da) reactive organic molecules (haptens) able to penetrate stratum corneum (SC), the top-most layer of the skin and covalently modify endogenous proteins are the most important contact allergens from a clinical point of view. Hapten expo- sure leads to activation of cutaneous dendritic cells (DCs), which migrate from the skin to the draining lymph nodes (LNs) where they present peptides (potential antigens) from hapten-modifed proteins to naïve T cells; thereby, activating the adaptive (acquired) immune system. Te naïve T cells that have recognized an antigen start to proliferate and diferentiate into antigen-specifc efector and memory T cells that circulate in the blood and lymphatic system3–5. Many aspects of the mechanisms underlying ACD are still unknown despite recent advances in understanding the role and interaction of diferent immune cells. For instance, although the hypothesis that haptens induce immune responses by modifying endogenous proteins was introduced by Landsteiner and Jacobs already in 19366 the identity of these modifed proteins has remained more or less unknown even until today. In particular, knowledge of the identity of hapten-protein conjugates seen as immunogenic by the immune sys- tem, i.e. that become antigens that activate naïve T cells, will increase our mechanistic understanding of contact allergy. In addition these hapten-protein conjugates could have potential as biomarkers for the development of better diagnostic tests for contact allergy and may also prove useful in the development of improved treatments. One previously unexplored approach to fnd potentially immunogenic hapten-modifed proteins would be to identify hapten-protein conjugates in the LNs afer topical administration. 1Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden. 2Department of Chemistry and Molecular Biology, Dermatochemistry, University of Gothenburg, Gothenburg, Sweden. Correspondence and requests for materials should be addressed to I.K. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:2895 | DOI:10.1038/s41598-018-21327-8 1 www.nature.com/scientificreports/ Te aim of the present study was to improve our understanding regarding the fate of a specifc hapten afer topical application and, in particular, to identify hapten-protein conjugates in LNs as these play a key role in activation of the adaptive immune system. Te hapten-protein conjugates (adducts) are most ofen formed by reactions between electrophilic haptens and nucleophilic side chains such as cysteines (thiols) and lysines (pri- mary amines) in skin proteins. Isothiocyanate (NCS) is an electrophilic functional group prone to react with both amines and thiols. Fluorescent isothiocyanates, e.g. fuorescein isothiocyanate (FITC), are due to their reac- tivity with proteins extensively used in various biological applications. In the present study mice were topically exposed to two fuorescent compounds, tetramethylrhodamine isothiocyanate (TRITC, sensitizer)7 and tetrae- thylrhodamine (Rhodamine B, non-sensitizer) and the distribution in skin and draining LNs was investigated. Microscopic examination of skin penetration of TRITC afer topical application showed that the most intense fuorescence signal was detected in SC. Excision and inspection of the draining LNs from mice sensitized to TRITC revealed fuorescence in DCs but also in T and B cell populations. No fuorescence was detected in LNs in mice treated with Rhodamine B. Detection of TRITC-modifed proteins isolated from LN cells, followed by identifcation of the most strongly TRITC-fuorescent protein, revealed TRITC bound to the N-terminal proline of the pleiotropic cytokine macrophage migration inhibitory factor (MIF). To the best of our knowledge, this is the frst time a hapten-protein conjugate has been identifed in local LNs afer topical application of the hapten. Results In the current project, the fuorescent skin sensitizer TRITC (Fig. 1) and the structurally similar compound Rhodamine B (Fig. 1), which is fuorescent but lacks the reactive NCS group of TRITC, were topically applied to the dorsum of mice ears for three consecutive days. Tereafer, the TRITC/Rhodamine B fuorescence in skin, draining LNs, LN cells and LN proteins was investigated. Sensitization assessment. Te skin sensitizing potential of Rhodamine B was investigated in the Local Lymph Node Assay (LLNA)8–10, which is the recommended OECD method for assessing the skin sensitizing potential of a compound. Rhodamine B was considered a non-sensitizer in the used test concentrations (up to 5% (w/v)) as no cell proliferation was induced in the LNs (Supporting Information, Table S1). Tis is in contrast to TRITC, which we have previously shown to be an extreme skin sensitizer in the LLNA with an EC3 value of 0.040%7. Visualization of fuorophore distribution in mouse ears. To study the distribution of TRITC and Rhodamine B in the skin afer topical in vivo exposure, sections of fuorophore-exposed mouse ears were inves- tigated using laser scanning confocal microscopy (LSCM). TRITC was detected mainly in SC, in hair follicles, in the matrix of the cartilage and to a minor extent in viable epidermis (Fig. 1a). To visualize the fuorescence from the control substance Rhodamine B, the laser power had to be increased 20 times compared to the settings for the TRITC sample. Te most intense Rhodamine B fuorescence was seen in hair follicles, while fuorescence in SC and cartilage was hardly detectable despite the increased laser power (Fig. 1b). Te discrepancy between the distributions of the two fuorophores in the skin is likely due to the NCS group’s ability to covalently modify biomacromolecules. We have previously shown in in vitro studies on human skin using LSCM and two photon microscopy that FITC, structurally similar to TRITC with corresponding skin sensitizing properties11, accumu- lated in SC while fuorescein, the non-sensitizing analogue lacking the NCS group, penetrated to deeper layers of epidermis12. Visualization of fuorophores in LNs and distribution in LN cells. To further investigate the distribu- tion of the fuorophores in mice, ear draining LNs were excised and analyzed with LSCM and fow cytometry. Te presence of TRITC was clearly visualized in LN sections (Fig. 1c), whereas the negative control Rhodamine B was not observed using LSCM (data not shown). Te number of LN cells was markedly increased in mice treated with TRITC and the proportion of fuorescent LN cells was estimated to on average 77% by fow cytometry (Table S2). Te number and fuorescence of LN cells in Rhodamine-exposed mice was the same as for vehicle-treated mice; hence corroborating the LSCM study in which no Rhodamine B fuorescence was detected in the LN section. Earlier skin painting studies with TRITC have identifed DCs as responsible for the accumulation of the fluorophore in LNs13–16. Indeed, our study shows that almost all DCs were TRITC positive (around 85%). However, our investigation analyzed diferent cell types and could show that T and B cells were the major cellular targets of the fuorophore (Fig. 2). While DCs constituted a minor part (2–3%) of the LN cell population, T-cells were the main cell population (around 90%) in the LNs of the TRITC exposed mice and two thirds of the T-cells were TRITC positive. B cells constituted about 10% of the LN cells and of these more than half were TRITC posi- tive. Tus, the high yield of TRITC positive cells in the LN is unlikely to solely depend on transport of haptenated proteins/peptides from the skin by LN immigrating DCs. Studies of other fuorescent isothiocyanates, by us17 and others18,19, have shown that in addition to the relatively slow cell-mediated transport of these compounds to the local LNs by DCs, they are also transported as free haptens or soluble hapten-protein complexes via the lymph and blood to the secondary lymphoid organs. In the LNs, these soluble haptens/hapten-protein conjugates might be captured by antigen-presenting cells such as resident DCs, macrophages, or B cells.